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Cacypcrative Agreement DEAC22-9OBCl4fj64.OOO

TheUnivdty OfMichigan Ann Arbor;Michigan

ContractDate: Jdy 1 1990 Anticipated Cmnpletion: June 30 1993 F-g for FY 1990 $50 OOO

ptincipd Xnvestigator: H. Scott Fogler

Project Manager: EdithAllison SartlesvilleRoject Office

Reporting Perid April I- June 30,1991 ____--. -_

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The objectives of this research are to elucidate and model bamrial transport in porous me&, to dete3mint the hpartance ofpolysaccharidts bridgh as a retentive m m m , and to identify key parameters that iuflucnce porons ~ P l ~ U & w b

This p j e c t has been subdivided into three tasks Task 1 i s the dtterminaton of the wth k h e t ~ s of theLRuconostoc bactmia and how they arc affccted by 1) the nutrient &., and 2) surface effects; Task 2 will quantify the importance of polysaccharide

productioa as a cell. retention mechanist#; and Task 3 is the elucidation of the rate of plysaccharide production and the combined effect that polysaccharide production and cell gmwth has upon plugging. I .

Batch growth experiments have been conducted fo determine the rate of dysaccharide production (dextran) during cell growth. In addition, culture studies have

L e n conducted for the verification of the grawth model.

The further verification of the two parameter model, as presented in the past 4uarttrly repwts, was the focus of these batch experiments. Three series o€ batch cU.ttm experiments, labeUd.as experimental series -20, X&21 and m - 2 3 , were conducted with varying yeast e x w t concenmtions and with no saccharides. These experiments were cwducted according to theproccdure detaildin carlicrquatterlyreports, with the exc tion that the itloculum comprised of cells directly cultivated from a stock cultt;tres purc ased fiom ATCC 14935, ie. new inoculum. Cells used in the past kinetic experiments werc originally from a stock of ATCC 14935 cells how eve^, &ey were continuously cultivated as lnoculum for the past kinetic eqerirzzenrs, ie. a cuztured inoculu#1.

The results from these experiments and the past p w t h experiments having yeast extract as the sale substrate axe presented in Table 1, As can be seen, the specific rate constants for the cells for the new inoculum are higher than the rates found from the past eqerimem using cultured cells as inoedum. '&is indicates that the cells used in the original experhem have undergone a phenotypic alteration, Thus, the Model developed could not be uedfkd and requires additional data. h futltrc experiments, the growth of the inoculum will bc confrolled by minimizing the number of celz transfers before use in kinetic 6kj)tirbents.

?tvo additional experimental series have been completed to determine the rate of ceUular production of dextran. The &t set of ex-ents consisted of two batch reactors containing 5 and 36 glL of sumse and 10 g/L of yeast extract in each. These mctors were inoculated with geJk that were wnthuously cultcrred as discused earlier. The second experimental Series consisted of cclk grown in feed containin6 5, 10,30 or 50 g sumsfi arui 10 g east cxtract/l. The inoculum used in the second smcs of experiments we.re new cells. In L th scrim the respective inwuIums were grown in a glucose-fructose faad for a

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24 hour cod, centrifuged, and then resuspended in thdr respective feed at the time of inoculahotr.

Table 1. Specific gmwth me as determined by batch cultures, Yeast E x m t a l Series

Extract conc. n o 9 =I1 KE16 KEN KE21 Ka23 0 Specific Growth Rate, p (hrl)

1 0.046

10 0.205 0.332 0.551 0.548 0.630 20 0,596 0.864 0.680 30 0.555 0.713 M e penmental Series ' KB 11, a n d 16 ,were inoculated with

5 ': 0.506 E3

*?$e con+inuously cui=

cellular dextran production wag determined by using the phenol-suIfuric assayl, while: cell counts where detemined by Culture Counter, as detailedin the lastrep- Figure 1 illustrates a growth curve and dwrtranpmduction curve for cells grown in a feed containing 36 g/L sucrose and 10 gL yeast extract, The curve demonstrates a lag between ductrmprcduction and cell growth, with cells owth alprays preceding polymer produrnon. This result was typical fur all batc F experiments, The difference in the duration of time before dextran prr;lduction and after cell p w t h indicates that dextran synthesis is a 9 llI product as typified b Garden? Type m products we known to be produced only w en cells reach maturity. &me the difference between the growth lag and the dextran synthesis lag is the time require for the cell to reach mamity. The comspmding growth rate, growth lag, dextran synthesis lag, and final dextran yields are presented in Table 2.

Two interesting results are presented by the data which should ?x discussed. The first being the ability ofthe cells to produce dextran aftermhbg maturity, Note that time required to reached mturity depends on the concentration of sucrase in the feed. However, at this t h e there does not seem to be any Cordarion between the length of this maturity period and the sucrose concentration. "he second result is &e Bifference in the cell growth rate and final dextran production yields when inoculated with continuously oultwed cells or new cells, The cell's growth rate for both types of inoculum were found to dvwd on the sum8e concentration in the fed; the rates for the continuously cultured cells were corn aratively lower. The lower cell growth rates were accarnpanid by an

and dextran synthesis are competing for sucrose for growth. Hence, even thou h the data from past experiments inoculated wth continuous cultured cells can not be us to model the cells growth, because the cell's have undergone a phenotypic alteration, the data does prwide us with information with respect to the ability of manipulating the feed and cells to control the cell's ability to d u c t polysaccharides, This mad dation will possibly enable UB to eventually &nce cell transport in pornus media YI y controling polysacchatidc production relative to cell production,

hcrca~e in the P mal dextran production yields. This mdt is expected since cell production

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Figure 1. The resulting cell and dextran concmtration when Leuconostoc cells are hoculated into a mediacontaining 36 g sucroseJL and 10 g yeast extract&.

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Table 2. CeUular growth rate, growth lag, dextran synthesis lag, and final dextran yields as a function of initial sucrose concentration.

Final Dextran

(a) (hr9 0 (ma)

I Sucrose l f i C Growth Conc. Ed h g Time

Rate (p.1 CbnC,

hocnlum-culm&cells

haulurn - new *US

5 0.684 1.2 '1.9 20 0.782 1.4 2.7 2200 36 0,746 1.1 2.6.. 2200

3 0,649 0.27 ' ' 2.73 138 15 0.832 0.9 * 2.1 522 30 0,907 0.89 1.6 700

' 50 0.782 0.5 1 4.5 ,, _,,,, . 1333

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1) Chaplin, M E and JJ?. Kennedy, Carbohydrate Analysis, IBR Press, Oxford E~&land 1986

2) Baile James E. andDavidF. OUis, Biochemical Engineering Fundamentals, MC & BW HiIl Book Co., 19'77